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Craig A. AspinwallAssociate Professor of Chemistry aspinwal@email.arizona.edu Old Chemistry 322 Phone: (520) 621-6338 Fax: (520) 621-8407 |
Honors
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Education and Appointments
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Research Interests
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Research Summary | |
| Development of Novel Methodologies for Investigations of Cellular Signal Transduction Analytical
Chemistry, Bioanalytical Chemistry, Biological Chemistry, Biophysics, Cell
Physiology, Sensors, Instrumentation
Investigations of the molecular interactions leading to cellular function (and dysfunction) are of paramount importance for a variety of biological research areas. Cellular function is regulated by an elegant series of chemical interactions between diverse classes of molecules including ions, phospholipids, neurotransmitters, nucleic acids and proteins. Chemical signals from both the intracellular and extracellular environments activate intracellular signaling through a variety of pathways including interaction with cell surface receptors, diffusion or transport across the cellular membrane, and interactions with ion channels.
Until recently, the ability to analyze these interactions at the single cell level has been limited by a dearth of analytical technologies capable of quantifying and qualifying such interactions in the cellular milieu. For example, cellular volumes are small (~10-12 L), the mass of the analyte is often low (<< 10-15 mol), and the environment is chemically complex. A variety of areas will benefit directly from development of novel technologies to address these types of analyses. The primary goal of our research is to develop technologies that will enable one to monitor the chemistry of life at a cellular and sub-cellular level and then to apply those methods to investigate problems of physiological significance, in our case chemical characterization of signal transduction pathways in the insulin-secreting pancreatic β-cell and cells from the parathyroid hormone releasing parathyroid gland. In order to dissect these processes, we are taking a multistage approach by developing technologies to measure the binding event, the ensuing intracellular signaling cascade and the resulting cellular function in order to directly correlate all of these events. Successful realization of our goals requires an interdisciplinary approach which includes biochemistry, analytical chemistry, materials chemistry and cell biology, along with other areas. In the area of analytical chemistry, we are developing a broad spectrum of analytical techniques and chemical sensors, ranging from microcolumn separations to fluorescence spectroscopy to electrochemistry and electrophysiology to ion channel-based chemical sensors that will provide a foundation upon which new biological problems can be solved. Some of our current approaches are outlined below. Analysis of Cellular Chemistry by Capillary Electrophoresis - Capillary electrophoresis (CE) has rapidly become an important method for rapid analysis of biological molecules. Our research focuses on the development of novel injection and detection technologies, along with new surface treatments to allow rapid, high-sensitivity CE analysis of complex samples. We are developing a new optical-gating instrument for online, rapid capillary separations based on the photolysis of caged fluorescent probes (Figure 2). In the photolytic injection scheme, optical gating is achieved by UV photolysis of a caged fluorescent label. Upon photolysis of the sample by 1-50 ms exposure to 351 nm light, the electron withdrawing caging groups are released resulting in fluorescent analytes that can then be separated and detected. Figure 2 shows a typical series of consecutive electropherograms of the amino acid neurotransmitter l-glutamate.
We are also investigating a series of new capillary coatings for analysis of proteins from complex matrices using the polymerizable phospholipids such as bis-SorbPC, (1,2'-bis[10-(2',4'-hexadienoyloxy)decanoyl]-sn-glyero-3 phosphocholine). Phospholipid bilayer coatings are comparable to surfactant coatings from a stability standpoint, but demonstrate higher recovery in CE separations based on the natural resistance to protein adsorption. Using polymerizable phospholipids we have formed coatings that are stable to surfactant, show little degradation over time and no lipid loss when coupled with ESI-MS. We have also begun to explore the idea of patterning varying surface functionalities, coupled to the lipid headgroups, in order to produce capillaries with arrays of chemical function. II. Development of biocompatible nanometer sized sensors for cellular analysis - Fluorescence imaging utilizing indicator dyes or fluorescent proteins has become routine but suffers from many limitations including toxicity, chemical stability and the possibility that the dye/label itself affects the chemical phenomena under investigation. The inability to detect chemically and biologically relevant analytes that do not possess intrinsic optical or electrochemical activity has imparted a further limitation for the more traditional methodologies. We are developing a novel class of nanometer sized chemical sensors prepared from chemically and environmentally stable polymerizable phospholipids with embedded biological signal transduction elements (Figure 3). The keys to this platform are the polymerized phospholipid membrane, which adds structural stability to the sensor, and the incorporation of biological signal transduction elements that serve to localize the sensors and/or transduce the signal by acting as the sensing element. In addition to providing stable sensors for intracellular analysis, continued development this sensor platform will provide stand alone nanometer sized chemical sensors that are tunable towards a range of applications, i.e. detection of chemical and biological warfare agents.
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Selected Publications | |
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